Whooping Cough or Pertussis is an acute, highly infectious respiratory disease caused by the Bordetella pertussis bacterium, a microorrganism formerly isolated by Bordet and Gengou in 1906 [Bordet, J. and O. Gengou. Ann Inst Pasteur (Paris), 1906. 20: p. 731-41]. Recently, the annual morbidity of infections throughout the world was estimated in 48.5 millions. The disease is particularly severe in children with less than six months of age, with 90% of the casualties being associated to this ethareal group (300,000-400,000) [Crowcroft, N. S., et al. Lancet Infect Dis, 2003. 3(7): p. 413-8].
Several vaccines are available against B. pertussis, distributed in two main groups according to their type: cellular vaccines, and, more recently, acellular vaccines. Vaccination has dramatically decreased the incidence of the disease, moving it from children towards teenager and adult populations. Several studies have shown teenagers as the major reservoir for B. pertussis and the main source for spreading of this disease among partially protected children. Therefore, whooping cough remains as an unsolved health problem, demanding the development of new vaccines for a better control of the epidemics and re-emergent outbreaks, and possibly to eradicate this disease in endemic regions [Cherry, J. D. Pediatrics, 2005. 115(5): p. 1422-7; Singh, M. and K. Lingappan, Chest, 2006. 130(5): p. 1547-53]. The Bordetella genera includes nine species, four of them being associated to infections in mammals (B. holmesii, B. bronchiseptica, B. parapertussis and B. pertussis), the last two being responsible for infections in humans [Mattoo, S., et al. Front Biosci, 2001. 6: p. E168-86]. Most of their virulence factors are regulated at transcriptional level by a two-components system denominated BvgA/S (Bordetella virulence genes Activator/Sensor) [Stibitz, S., et al. Nature, 1989. 338(6212): p. 266-9]. Among them, the most relevant factors are the Pertussis toxins (PT), the tracheal colonization factor, adenylate cyclase; and adhesins filamentous Phytohemagglutinin (PHA), Fimbriae (Fim) and Pertactin (Prn), the latter being the focus of the present invention.
Prn is an outer membrane protein belonging to the family of type V autotransporter proteins. It is characterized by catalyzing its own transportation through the bacterial outer membrane [Henderson, I.R. Trends Microbiol, 2000. 8(12): p. 534-5]. The mature Prn is a protein of 68 kDa in B. bronchiseptica [Henderson, I.R. Infect Immun, 2001. 69(3): p. 1231-43.], 69 kDa in B. pertussis [Charles, I. G., et al. Proc Natl Acad Sci USA, 1989. 86(10): p. 3554-8] and 70 kDa in B. parapertussis [Li, L. J., et al. Mol Microbiol, 1991. 5(2): p. 409-17], respectively. Its structure consists on 16 paralel strands forming a β helix and a transversal section in V form [Emsley, P., et al. Nature, 1996. 381(6577): p. 90-2.]. Numerous loops protrude from this helicoidal core. One of them is the Arg-Gly-Asp triplete (RGD), a motif associated to tissue adherence [Leininger, E., et al. Infect Immun, 1992. 60(6): p. 2380-5; Emsley, P., et al. Nature, 1996. 381(6577): p. 90-2]. The presence of this motif and numerous proline-rich regions are related to Prn functions during adhesion. Experiments have shown that the Prn can mediate adhesion to cells of the respiratory epithelium [Everest, P., et al. Microbiology, 1996. 142 (Pt 11): p. 3261-8]. Nevertheless, assays on the inhibition by human sera of B. pertussis adhesion to A549 cultured cells (alveolar human epithelium) did not evidence Prn as a crucial element during that process under the tested conditions [Rodriguez, M. E., et al. FEMS Immunol Med Microbiol, 2006. 46(1): p. 39-47].
The Prn protein is part of acellular vaccines composed of three or more components. Acellualr vaccines can be composed of: 1) one component of PT, 2) two components: PT and PHA, 3) three components: PT, PHA and Prn, and 4) five components, including the three components previously mentioned and also the Fimbriae 2 (Fim2) and Fimbriae 3 (Fim3) proteins. In humans, the levels of the anti-Prn, anti-Fim2 and anti-PT antibodies correlate with protection levels against the disease [Cherry, J. D., et al. Vaccine, 1998. 16(20): p. 1901-6; Storsaeter, J., et al. Vaccine, 2003. 21(25-26): p. 3542-9].
The active immunization with Prn of B. pertussis and B. bronchiseptica induces a specific antibody response against Prn, confering protection in different animal models [Charles, I. G., et al. Eur J Immunol, 1991. 21(5): p. 1147-53; Roberts, M., et al. Vaccine, 1992. 10(1): p. 43-8]. Similarly, the passive administration of anti-Prn monoclonal antibodies (MAbs) protected mice in the model of respiratory challenge [King, A. J., et al. Microbiology, 2001. 147(Pt 11): p. 2885-95]. Protection levels in mice subjected to the intranasal challenge assay (INCA) were increased by adding Prn to vaccines containing PT and PHA [Guiso, N., et al. Vaccine, 1999. 17(19): p. 2366-76]. It has been recently shown that Prn is the only component of acellular vaccines which generates an antibody response of such a level that correlates to the opsonophagocytic activity [Hellwig, S. M., et al. J Infect Dis, 2003. 188(5): p. 738-42]. In spite of efficacious vaccines and the well established vaccination programs available, whooping cough is still endemic in regions of America, Europe and Asia, being considered as a re-emergent disease [Raguckas, S. E., et al. Pharmacotherapy, 2007. 27(1): p. 41-52]. One of the hypotheses trying to explain this phenomenon is based on the loss of efficacy, due to appearance of resistant strains [Mooi, F. R et al. Emerg Infect Dis, 2001. 7(3 Suppl): p. 526-8]. Prn is one of the most polymorphic proteins in B. pertussis. It contains two variable regions designated as region 1 (R1) and 2 (R2), respectively, with repetitive amino acid sequences rich in proline Gly-Gly-X-X-Pro (GGXXP) (SEQ. ID. No. 7) and Pro-Gln-Pro (PQP) motifs. The R1 region is located in the protruding loop proximal to the aminoterminal sequence (N-terminal) and near to the RGD motif, while the R2 region is located near to the carboxyl terminal end (C-terminal) [Hijnen, M., et al. Infect Immun, 2004. 72(7): p. 3716-23]. Up to 12 different variants of Prn (Prn1, Prn2, Prn3 . . . Prn12) have been identified in B. pertussis, as shown in the database of the National Center for Biotechnology Information of the United States of America (NCBI). Strains bearing the Prn1, Prn2 and Prn3 are distributed worldwide. Numerous strain characterization studies, either retrospective or of strains currently circulating, were carried out in American, European, Asian and Australian regions and showed a tendency towards a progressive persistence of Prn2 strains over Prn1 strains, the Prn2 strains predominating in most of the countries studied [Mooi, F. R., et al. Infect Immun, 1998. 66(2): p. 670-5; Cassiday, P et al. J Infect Dis, 2000. 182(5): p. 1402-8; Weber, C. et al. J Clin Microbiol, 2001. 39(12): p. 4396-403; Hallander, H. O., et al. J Clin Microbiol, 2005. 43(6): p. 2856-65; van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p. 2837-43; Byrne, S, et al. BMC Infect Dis, 2006. 6: p. 53].
Current differences in the amino acid sequence of Prn between cellular (DPTc) or acellular (DPTa) vaccines and circulating strains is one of the factors supporting the hypothesis of the efficacy loss of vaccines available, due to the appearance of new strains. Studies in populations vaccinated with DPTc or (DPTa), and non-vaccinated populations, in Netherlands and Italy indicated that these types of vaccines protect better against circulating strains similar to the vaccine strain [Mooi, F. R., et al. Infect Immun, 1998. 66(2): p. 670-5; Mastrantonio, P., et al. Microbiology, 1999. 145 (Pt 8): p. 2069-75]. In agreement with these findings, it was shown in the mice model that vaccination with DPTc differentially protects against strains bearing Prn1 and Prn2, indicating that changes in the Prn R1 region can confer resistance levels [King, A. J., et al. Microbiology, 2001. 147(Pt 11): p. 2885-95]. However, massive studies stratifying B. pertussis strains according to country of origin, vaccination status, and type of vaccines (DPTc and DPTa), did not show significant differences in the frequencies of prn, ptxC, ptxA or tcfA2 alleles for circulating strains and vaccination programs [van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p. 2837-43].
The high prevalence of Prn2 strains in many countries is indicative of the favored transmission of these strains by means still unraveled, although the findings mentioned above hardly link the origin of new variants to vaccination. Remarkably, in the above mentioned study [van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p. 2837-43], the three clinically isolated strains bearing allelles similar to that of the vaccines used were found only in non-vaccinated children. Either casual or not, it suggests that Prn1 strains are favored in niches devoid of specific immunity. On the other hand, the recent identification of a phage infecting Bordetella (BPP-1) by using Prn as primary receptor, suggested that variations in this protein might be triggered by selective pressures other than those imposed by the immune system [Liu, M., et al. Science, 2002. 295(5562): p. 2091-4]. The possible influences of both phenomena, together with other unknown factors leading to harmonized variations in B. pertussis, are not excluded.
The evolution of Pertussis epidemiology has been simulated by a mathematical model, integrating the incidence of the disease and the pathogen's transmission independently [Aguas, R., et al. Lancet Infect Dis, 2006. 6(2): p. 112-7]. This model predicts that regular boosting doses would not be capable of eliminating the severity grades of the disease, observed in current epidemics. It is highly probable that this should be caused by the short lifespan of the protection conferred by the available acellular vaccines (4-12 years), and also the variability of the immune response and the different types of vaccines. This model predicts as the most optimistic scenario that where vaccines could generate an immunity superior to the natural one, a paradigm still unreached by the cellular and acellular vaccines available.
The main purpose of the present invention resides on the contribution to develop more efficacious acellular vaccines against Whooping Cough. The main work preceding the present invention were based on administering immunogenic preparations obtained by mixing Prn proteins (Nicole Guiso et al., WO 01/90143 A2 y US 2006/0008474 A1) or synthetic peptides of the Prn R1 region (Frederik Mooi et al., WO 02/00695 A2). Therefore, the development of more efficacious acellular vaccines is an important problem to prevent Whooping Cough.